RESUMO
PHARMACOLOGY RELEVANCE: Baccharis trinervis (Lam, Persoon) leaves are used in the traditional medicine for the treatment of high fevers, edema, inflammation, sores and muscle cramps, snakebites and as antiseptic. AIM OF THE STUDY: To investigate the cytotoxic, genotoxic, and mutagenic effects of extracts and fractions of B. trinervis from Brazil and Colombia in Chinese Hamster Ovary (CHO) cells, and to examine the mutagenic activity in Salmonella typhimurium. MATERIAL AND METHODS: Aqueous extracts (AE) of aerial parts of B. trinervis from Brazil (B) and Colombia (C) were fractioned in ethyl acetate fraction (EAF), butanol extract (BF), and aqueous residue fraction (ARF). Qualitative chemical screening and determination of total flavonoid content were made. Identification of chemical constituents was performed by High Performance Liquid Chromatography (HPLC) and High Resolution Mass Spectrometry (HRMS). For the in vitro tests, CHO cells were treated for 3h with extracts and fractions. The cytotoxic activity was evaluated by clonal survival and 3-(4.5-dimethylthiazole-2-yl)-2.5-biphenyl tetrazolium bromide reduction assay (MTT). Genotoxic and mutagenic effects were evaluated by the alkaline comet assay and Cytokinesis-blockage micronucleus test (CBMN), respectively. Additionally, Salmonella/microsome assay was carried out to determinate the mutagenic effects in EAF from Brazil and Colombia. RESULTS: Phytochemical analyses indicated the presence of saponins and flavonoids. AE and EAF were the samples with the highest quantity of total flavonoids. HPLC showed the presence of luteolin only in AEC, and caffeic acid, ellagic acid, rosmarinic acid, and rutin were identified in AEB and AEC (AEC>AEB). The HRMS in positive mode of EAFB and EAFC showed presence of two carboxylic acids, coumarin, and two terpenoids. In addition, were identified one terpenoid and two carboxylic acids in AE, BF and ARF of B. trinervis from both countries in negative mode. Dose-dependent cytotoxic effects were observed in CHO cells treated with B. trinervis extracts and fractions by using clonal survival and MTT at concentrations higher than 0.05mg/mL. All the extracts and fractions induced DNA strand breaks in CHO cells with dose-dependent response, mostly EAFB and EAFC. The EAF from Brazil and Colombia showed mutagenic effect at 0.5mg/mL, while the other fractions did not show a significant difference in relation to the control. No mutagenic effects were found in EAF from both countries by the Salmonella/microsome assay. CONCLUSIONS: Cytotoxic and genotoxic effects were demonstrated in all extracts and fractions used, although only EAF showed mutagenic effects by CBMN, but not by Salmonella/microsome assay. Our results suggest that flavonoids, phenylpropanoids, coumarins, and diterpenes may be responsible for the cytotoxic, genotoxic and mutagenic effects observed.
Assuntos
Baccharis/química , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Flavonoides/análise , Mutagênicos/farmacologia , Extratos Vegetais/toxicidade , Animais , Brasil , Colômbia , Ensaio Cometa , Cricetulus , Relação Dose-Resposta a Droga , Testes para Micronúcleos , Microssomos/efeitos dos fármacos , Folhas de Planta/químicaRESUMO
Abstract Baccharis is an Asteraceae genus of flowering plants, which has about 340 to 400 species, ranging from the Southern United States to the Southern extreme of Argentina and Chile through Central America and the Caribbean regions. The species Baccharis trinervis is a native shrub from Mexico, Central America and throughout South America. In Costa Rica, this species is commonly known as alcotán and the fresh leaves are used as a poultice on wounds and ulcers. The objective of the present research was to characterize the chemical composition of seven hydrodistilled essential oils of diverse morphological parts of B. trinervis. For this, samples were obtained from three locations in Costa Rica and standard laboratory analyses were followed. The essential oils were analyzed by capillary gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) using the retention indices on a 5 % phenyl/dimethylpolysiloxane fused silica column in addition to mass spectral fragmentation patterns, which allowed the identification of 268 compounds. The essential oils consisted mainly of terpenoids (92.3 to 97.8 %). The major constituents from the leaf oils were caryophyllene oxide (0.1-22.5 %), viridiflorol (8.8-21.0 %), germacrene D (0.5-19.1 %), germacrene B (0.2-16.0 %), β-caryophyllene (3.5-9.1 %), spathulenol (0.1-8.3 %), δ-3-carene (2.0-6.8 %), and α-pinene (2.5-5.9 %). The flower oil consisted mainly of globulol (0-24 %), β-caryophyllene (9.5-17.1 %), cismuurola-4(14), 5-diene (traces-13.7 %), germacrene D (4.3-9.9 %), bicyclogermacrene (5.9-8.3 %), ar-curcumene (0-8.0 %), spathulenol (4.3-4.8 %), caryophyllene oxide (3.1-4.7 %), and viridiflorol (0.3-4.7 %). The major components of the branch oil were germacrene B (1.4-18.7 %), germacrene D (14.7-15.6 %), β-caryophyllene (10.1-12.4 %), viridiflorol (0-11.5 %), globulol (0.6-11.3 %), δ-3-carene (4.1-8.1 %), β-phellandrene (1.5-6.5 %), and bicyclogermacrene (3.6-4.9 %). The essential oil composition differed markedly from that of previously studied oils of plants growing in Brazil, which contain two characteristic stereoisomeric methyl dec-2-en4,6-diynoate compounds not detected in this study. This is the first report about the chemical composition of the essential oils obtained from this species growing wild in Costa Rica.
Resumen Baccharis (Asteraceae) es un género de plantas con flor que consta de 340 a 400 especies que habitan desde el sur de EE. UU. hasta Argentina y Chile, incluyendo América Central y varias islas del Caribe. Baccharis trinervis es un arbusto nativo de México, América Central y América del Sur. En Costa Rica, esta especie se conoce popularmente como alcotán y las hojas frescas se utilizan en forma de cataplasma para curar heridas y úlceras. El objetivo del presente estudio fue el de identificar los constituyentes químicos de los aceites esenciales obtenidos de diferentes partes morfológicas de B. trinervis en tres localidades de Costa Rica, obtenidos mediante el método de hidrodestilación. Se analizó la composición química de los aceites por cromatografía capilar de gases con detector de ionización de flama (GCFID) y cromatografía de gases acoplada a un detector de masas (GC-MS), utilizando los índices de retención en una columna tipo DB-5 y los patrones de fragmentación, lo cual permitió la identificación de 268 constituyentes. Los siete aceites están constituidos principalmente por terpenoides (92.3 a 97.8 %). Los compuestos mayoritarios de los aceites de las hojas se identificaron como óxido de cariofileno (0.1-22.5 %), viridiflorol (8.8-21.0 %), germacreno D (0.5-19.1 %), germacreno B (0.2-16.0 %), β-cariofileno (3.5-9.1 %), espatulenol (0.1-8.3 %), δ-3-careno (2.06.8 %), α-pineno (2.5-5.9 %), biciclogermacreno (0-4.4 %), δ-cadineno (0.8-3.4 %), β-elemeno (0.8-3.0 %), limoneno (0.9-2.9 %) y β-pineno (1.3-2.5 %). Los aceites de las flores contienen principalmente globulol (0-24 %), β-cariofileno (9.5-17.1 %), cis-muurola-4(14),5-dieno (t-13.7 %), germacreno D (4.3-9.9 %), biciclogermacreno (5.9-8.3 %), ar-curcumeno (0-8.0 %), espatulenol (4.3-4.8 %), óxido de cariofileno (3.1-4.7 %), viridiflorol (0.3-4.7 %), β-elemeno (2.5-4.3 %), germacreno B (1.8-4.3 %), γ-cadineno (0.32.5 %), δ-3-careno (0.9-2.2 %) y α-humuleno (1.8-2.2 %). Los constituyentes mayoritarios del aceite de las ramitas fueron: germacreno B (1.4-18.7 %), germacreno D (14.7-15.6 %), β-cariofileno (10.1-12.4 %), viridiflorol (0-11.5 %), globulol (0.6-11.3 %), δ-3-careno (4.1-8.1 %), β-felandreno (1.5-6.5 %), biciclogermacreno (3.6-4.9 %), δ-cadineno (3.5-4.3 %), β-copaeno (0.6-2.8 %), β-elemeno (1.3-2.7 %) y γ- muuroleno (1.6-2.6 %). Los aceites estudiados presentan una composición compleja y se diferencian de los aceites obtenidos de la misma especie que crece en Brasil por la ausencia de los compuestos isoméricos diacetilénicos dec-2-en-4,6-diinoato de metilo(Z y E). También se diferencian de los aceites de las plantas de Brasil y Venezuela por la presencia de sesquiterpenoides de la familia de los guayanos, en especial por cantidades apreciables de viridiflorol, globulol y espatulenol.